Process for the preparation of L-amino acids by fermentation and nucleotide sequences coding for the accDA gene

ABSTRACT

The invention relates to nucleotide sequences coding for the accDA gene and to a process for the preparation of L-amino acids, especially L-lysine, by fermentation using corynebacteria in which the accDA gene is amplified.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119 to Germanapplication 199 24 365.4, filed on May 27, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides nucleotide sequences coding for the accDA geneand a process for the preparation of L-amino acids, especially L-lysine,by fermentation using corynebacteria in which the accDA gene isamplified.

2. Background Information

L-Amino acids, especially L-lysine, are used in animal nutrition, inhuman medicine and in the pharmaceutical industry.

It is known that these amino acids are prepared by the fermentation ofstrains of corynebacteria, especially Corynebacterium glutamicum.Because of their great importance, attempts are constantly being made toimprove the preparative processes. Improvements to the processes mayrelate to measures involving the fermentation technology, e.g. stirringand oxygen supply, or the composition of the nutrient media, e.g. thesugar concentration during fermentation, or the work-up to the productform, e.g. by ion exchange chromatography, or the intrinsic productivitycharacteristics of the microorganism itself.

The productivity characteristics of these microorganisms are improved byusing methods of mutagenesis, selection and mutant choice to givestrains which are resistant to antimetabolites, e.g. the lysine analogS-(2-aminoethyl)cysteine, or auxotrophic for amino acids of regulatorysignificance, and produce L-amino acids.

Methods of recombinant DNA technology have also been used for some yearsin order to improve L-amino acid-producing strains of Corynebacterium byamplifying individual amino acid biosynthesis genes and studying theeffect on L-lysine production. Surveys of this subject have beenpublished inter alia by Kinoshita (“Glutamic Acid Bacteria” in: Biologyof Industrial Microorganisms, Demain and Solomon (Eds.), BenjaminCummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)),Eggeling (Amino Acids 6, 261-272 (1994)), Jetten and Sinskey (CriticalReviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals ofthe New York Academy of Science 782, 25-39 (1996)).

The enzyme acetyl-CoA carboxylase catalyzes the carboxylation ofacetyl-CoA to malonyl-CoA. The enzyme from Escherichia coli consists offour subunits. The accB gene codes for biotin carboxyl carrier protein,the accC gene for biotin carboxylase and the accA and accD genes fortranscarboxylase (Cronan and Rock, Biosynthesis of Membrane Lipids, in:Escherichia coli and Salmonella typhimurium (ed. F. C. Neidhardt), 1996,pp. 612-636, American Society for Microbiology). Because of the propertyof the enzyme to carboxylate acyl groups in the form of acyl-CoA, it isalso called acyl-CoA carboxylase.

The nucleotide sequence of the accBC gene from Corynebacteriumglutamicum has been determined by Jäger et al. (Archives of Microbiology166, 76-82 (1996)) and is generally available from the data bank of theEuropean Molecular Biologies Laboratories (EMBL, Heidelberg, Germany)under accession number U35023. The accBC gene codes for a subunit ofacetyl-CoA carboxylase which carries a biotin carboxyl carrier proteindomain and a biotin carboxylase domain.

OBJECT OF THE INVENTION SUMMARY OF THE INVENTION

The object which the inventors set themselves was to provide novelprocedures for the improved preparation of L-amino acids, especiallyL-lysine, by fermentation.

DESCRIPTION OF THE INVENTION

L-Amino acids are used in animal nutrition, in human medicine and in thepharmaceutical industry. It is therefore of general interest to providenovel improved processes for the preparation of L-amino acids.

When L-lysine or lysine is mentioned in the following text, it isunderstood as meaning not only the base but also the salts, e.g. lysinemonohydrochloride or lysine sulfate.

The invention provides a preferably recombinant DNA originating fromCorynebacterium which is capable of replication in coryneformmicroorganisms and which at least contains the nucleotide sequencecoding for the accDA gene shown in SEQ ID No. 1.

The invention also provides a DNA capable of replication, as claimed inclaim 1, with:

(i) the nucleotide sequence shown in SEQ ID No. 1, or

(ii) at least one sequence corresponding to the sequence (i) within theregion of degeneracy of the genetic code, or

(iii) at least one sequence hybridizing with the sequence complementaryto the sequence (i) or (ii), and optionally

(vi) [sic] neutral sense mutations in (i).

The invention also provides coryneform microorganisms, especially of thegenus Corynebacterium, transformed by the introduction of said DNAcapable of replication.

The invention further relates to a process for the preparation ofL-amino acids by fermentation using corynebacteria which, in particular,already produce the L-amino acids and in which the nucleotide sequencescoding for the accDA gene are amplified and, in particular,overexpressed.

Finally, the invention also provides a process for the amplification ofacyl-CoA carboxylase in corynebacteria by joint overexpression of thenovel accDA gene according to the invention and the known accBC gene.

In this context the term “amplification” describes the increase in theintracellular activity, in a microorganism, of one or more enzymes whichare coded for by the appropriate DNA, for example by increasing the copynumber of the gene(s), using a strong promoter or using a gene codingfor an appropriate enzyme with a high activity, and optionally combiningthese measures.

The microorganisms which the present invention provides can produceL-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch or cellulose or from glycerol and ethanol. Saidmicroorganisms can be representatives of corynebacteria, especially ofthe genus Corynebacterium. The species Corynebacterium glutamicum may bementioned in particular in the genus Corynebacterium, being known tothose skilled in the art for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, especially of the speciesCorynebacterium glutamicum for example, are the known wild-type strains:

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium thermoaminogenes FERM BP-1539

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020 and L-amino acid-producing mutantsor strains prepared therefrom, for example:

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P 1712

Corynebacterium glutamicum FERM-P 6463 and

Corynebacterium glutamicum FERM-P 6464

The inventors have succeeded in isolating the novel accDA gene from C.glutamicum. The accDA gene or other genes are isolated from C.glutamicum by first constructing a gene library of this microrganism[sic] in E. coli. The construction of gene libraries is documented ingenerally well-known textbooks and handbooks. Examples which may bementioned are the textbook by Winnacker entitled From Genes to Clones,Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990)or the handbook by Sambrook et al. entitled Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A verywell-known gene library is that of the E. coli K-12 strain W3110constructed by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors.Bathe et al. (Molecular and General Genetics 252, 255-265 (1996))describe a gene library of C. glutamicum ATCC13032 constructed usingcosmid vector SuperCos I (Wahl et al., Proceedings of the NationalAcademy of Sciences USA 84, 2160-2164 (1987)) in the E. coli K-12 strainNM554 (Raleigh et al., Nucleic Acids Research 16, 1563-1575 (1988)).Börmann et al. (Molecular Microbiology 6(3), 317-326) in turn describe agene library of C. glutamicum ATCC13032 using cosmid pHC79 (Hohn andCollins, Gene 11, 291-298 (1980)). A gene library of C. glutamicum in E.coli can also be constructed using plasmids like pBR322 (Bolivar, LifeSciences 25, 807-818 (1979)) or pUC9 (Viera et al., Gene 19, 259-268(1982)). Restriction- and recombination-defective E. coli strains areparticularly suitable hosts, an example being the strain DH5αmcrdescribed by Grant et al. (Proceedings of the National Academy ofSciences USA 87, 4645-4649 (1990)). The long DNA fragments cloned usingcosmids can then in turn be subcloned into common vectors suitable forsequencing, and subsequently sequenced, e.g. as described by Sanger etal. (Proceedings of the National [sic] of Sciences of the United Statesof America [sic] USA 74, 5463-5467 (1977)).

The novel DNA sequence from C. glutamicum coding for the accDA gene wasobtained in this way and, as SEQ ID No. 1, is part of the presentinvention. The coding region (cds) of the accDA gene is shown in SEQ IDNo. 2. The amino acid sequence of the corresponding protein was alsoderived from the present DNA sequence by the methods described above.The resulting amino acid sequence of the accDA gene product is shown inSEQ ID No. 3.

Coding DNA sequences which result from SEQ ID No. 1 due to thedegeneracy of the genetic code are also part of the invention.Similarly, DNA sequences which hybridize with SEQ ID No. 1 or sectionsof SEQ ID No. 1 are part of the invention. Furthermore, conservativeamino acid exchanges, e.g. the exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are known to those skilledin the art as sense mutations, which do not cause a fundamental changein the activity of the protein, i.e. they are neutral. It is also knownthat changes at the N and/or C terminus of a protein do notsubstantially impair its function or can even stabilize it. Thoseskilled in the art will find information on this subject inter alia inBen-Bassat et al. (Journal of Bacteriology 169, 751-757 (1987)), O'Reganet al. (Gene 77, 237-251 (1989)), Sahin-Toth et al. (Protein Sciences 3,240-247 (1994)), Hochuli et al. (Bio/Technology 6, 1321-1325 (1988)) andwell-known textbooks on genetics and molecular biology. Amino acidsequences which correspondingly result from SEQ ID No. 3 are also partof the invention.

The inventors have found that overexpression of the accDA genes incorynebacteria improves L-lysine production.

An overexpression can be achieved by increasing the copy number of theappropriate genes or mutating the promoter and regulatory region or theribosome binding site located upstream from the structural gene.Expression cassettes incorporated upstream from the structural gene workin the same way. Inducible promoters additionally make it possible toincrease the expression in the course of L-lysine production byfermentation. Measures for prolonging the life of the mRNA also improvethe expression. Furthermore, the enzyme activity is also enhanced bypreventing the degradation of the enzyme protein. The genes or geneconstructs can either be located in plasmids of variable copy number orbe integrated and amplified in the chromosome. Alternatively, it is alsopossible to achieve an overexpression of the genes in question bychanging the composition of the media and the culture technique.

Those skilled in the art will find appropriate instructions inter aliain Martin et al. (Bio/Technology 5, 137-146 (1987)), Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), EP 0 472 869,U.S. Pat. No. 4,601,893, Schwarzer and Pühler (Bio/Technology 9, 84-87(1991)), Reinscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007(1993)), patent application WO 96/15246, Malumbres et al. (Gene 134,15-24 (1993)), Japanese Offenlegungsschrift JP-A-10-229891, Jensen andHammer (Biotechnology and Bioengineering 58, 191-195 (1998)), Makrides(Microbiological Reviews 60, 512-538 (1996)) and well-known textbooks ongenetics and molecular biology.

An example of a plasmid by means of which the accDA gene can beoverexpressed is pZ1accDA (FIG. 1), which is contained in the strainMH20-22B/pZlaccDA. Plasmid pZ1accDA is an E. coli-C. glutamicum shuttlevector which carries the accDA gene and is based on plasmid pZ1 (Menkelet al., Applied and Environmental Microbiology 55(3), 684-688 (1989)).Other plasmid vectors capable of replication in C. glutamicum, e.g.pEKEx1 (Eikmanns et al., Gene 102, 93-98 (1991)) or pZ8-1 (EP 0 375889), can be used in the same way.

The inventors have also found that overexpression of the known accBCgene in addition to the novel accDA gene according to the invention incorynebacteria improves acyl-CoA carboxylase production. An example of aplasmid by means of which the accDA gene and the accBC gene can bejointly overexpressed is pEK0accBCaccDA (FIG. 2). Plasmid pEK0accBCaccDAis an E. coli—C. glutamicum shuttle vector which carries the accBC andaccDA genes and is based on plasmid pEK0 (Eikmanns et al., Gene 102,93-98 (1991)). Other plasmid vectors capable of replication in C.glutamicum, e.g. pEKEx1 (Eikmanns et al., Gene 102, 93-35 98 (1991)) orpZ8-1 (EP 0 375 889), can be used in the same way.

In addition, it can be advantageous for L-amino acid production tooverexpress not only the accDA gene but also one or more enzymes of thebiosynthetic pathway. Thus it is possible, for example for thepreparation of L-lysine,

simultaneously to overexpress the dapA gene coding fordihydrodipicolinate synthase (EP-B 0 197 335), or

simultaneously to amplify a DNA fragment conferringS-(2-aminoethyl)cysteine resistance (EP-A 0 088 166).

Furthermore, it can be advantageous for the production of L-amino acids,especially L-lysine, to switch off undesirable secondary reactions aswell as overexpress the accDA gene (Nakayama: “Breeding of Amino AcidProducing Micro-organisms” in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention can be cultivatedfor L-lysine production continuously or discontinuously by the batchprocess, the fed batch process or the repeated fed batch process. Asummary of known cultivation methods is provided in the textbook byChmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik(Bioprocess Technology 1. Introduction to Bioengineering) (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Bioreactors and PeripheralEquipment) (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of theparticular strains. Descriptions of culture media for variousmicroorganisms can be found in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981). Carbon sources which can be used aresugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose,maltose, molasses, starch and cellulose, oils and fats, e.g. soya oil,sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmiticacid, stearic acid and linoleic acid, alcohols, e.g. glycerol andethanol, and organic acids, e.g. acetic acid. These substances can beused individually or as a mixture. Nitrogen sources which can be usedare organic nitrogen-containing compounds such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soybean flourand urea, or inorganic compounds such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.The nitrogen sources can be used individually or as a mixture.Phosphorus sources which can be used are phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium salts. The culture medium must also contain metalsalts, e.g. magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth-promoting substances such as aminoacids and vitamins can be used in addition to the substances mentionedabove. Suitable precursors can also be added to the culture medium. Saidfeed materials can be added to the culture all at once or fed inappropriately during cultivation.

The pH of the culture is controlled by the appropriate use of basiccompounds such as sodium hydroxide, potassium hydroxide, ammonia oraqueous ammonia, or acid compounds such as phosphoric acid or sulfuricacid. Foaming can be controlled using antifoams such as fatty acidpolyglycol esters. The stability of plasmids can be maintained by addingsuitable selectively acting substances, e.g. antibiotics, to the medium.Aerobic conditions are maintained by introducing oxygen oroxygen-containing gaseous mixtures, e.g. air, into the culture. Thetemperature of the culture is normally 20° C. to 45° C. and preferably25° C. to 40° C. The culture is continued until formation of the desiredL-amino acid has reached a maximum. This objective is normally achievedwithin 10 hours to 160 hours.

L-Lysine can be analyzed takes place [sic] by means of anion exchangechromatography followed by ninhydrin derivatization, as described bySpackman et al. (Analytical Chemistry 30, 1190 (1958)).

The following microorganisms have been deposited in the DeutscheSammlung für Mikrorganismen [sic] und Zellkulturen (German Collection ofMicrorganisms [sic] and Cell Cultures (DSMZ), Brunswick, Germany) underthe terms of the Budapest Treaty:

Corynebacterium glutamicum strain DSM5715/pZ1accDA as DSM12785

Corynebacterium glutamicum strain DSM5715/pEK0accBCaccDA as DSM12787

The process according to the invention is used for the preparation ofL-amino acids, especially L-aspartic acid, L-asparagine, L-homoserine,L-threonine, L-isoleucine and L-methionine, by the fermentation ofcorynebacteria. It is used particularly for the preparation of L-lysine.

EXAMPLES DETAILED DESCRIPTION OF THE INVENTION

The present invention is illustrated in greater detail below with theaid of Examples.

Example 1

Cloning and Sequencing of the accDA Gene

A gene library of C. glutamicum ATCC13032 was constructed using cosmidpHC79 (Hohn and Collins, Gene 11, 291-298 (1980)), as described byBörmann et al. (Molecular Microbiology 6(3), 317-326).

A chosen cosmid was digested with the restriction enzymes EcoRI and XhoIas instructed by the manufacturer of these restriction enzymes(Boehringer Mannheim). The DNA fragments formed were mixed with vectorpUC18 (Norrander et al., Gene 26, 101-106 (1983)), which had also beentreated with the restriction enzymes EcoRI and XhoI, and, aftertreatment with T4 DNA ligase, were cloned into the E. coli strainDH5αmcr (Grant et al., Proceedings of the National Academy of SciencesUSA 87, 4645-4645 [sic] (1990)), as described by Sambrook et al.(Molecular Cloning, a Laboratory Manual (1989), Cold Spring HarborLaboratories). The transformants were selected on LB agar containing 50μg/ml of ampicillin, as described by Sambrook et al. (Molecular Cloning,a Laboratory Manual (1989), Cold Spring Harbor Laboratories). PlasmidDNA was isolated from a transformant and called pUCaccDA. Subclones werethen prepared, via exonuclease III digestion, using the kit(Erase-a-Base) provided for this purpose by Promega (Heidelberg,Germany). Said subclones were sequenced by the dideoxy chain terminationmethod of Sanger et al. (Proceedings of the National Academy of SciencesUSA 74, 5463-5467 (1977)). This was done using the Auto-Read SequencingKit (Amersham Pharmacia Biotech, Uppsala, Sweden). Gel electrophoreticanalysis was carried out with the automatic laser fluorescence (A.L.F.)sequencer from Amersham Pharmacia Biotech (Uppsala, Sweden). Thenucleotide sequence obtained was analyzed with the HUSAR softwarepackage (Release 4.0, EMBL, Heidelberg, Germany). The nucleotidesequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequenceshowed an open reading frame of 1473 base pairs, which was called theaccDA gene. The accDA gene from C. glutamicum codes for a polypeptide of484 amino acids.

Example 2

Expression of the accDA Gene in Corynebacterium Glutamicum

The accDA gene was subcloned into vector pZ1 (Menkel et al., Applied andEnvironmental Microbiology 55, 684-688 (1989)) for expression in C.glutamicum. This was done by cleaving plasmid pUCaccDA (cf. Example 1)with the restriction enzyme ClaI. The resulting 1.6 kb fragment wasisolated as described in Example 1, treated with Klenow polymerase andalkaline phosphatase and used for ligation to pZ1, said vector havingbeen linearized with ScaI beforehand. The ligation mixture was used totransform E. coli DH5αmcr (Grant et al., Proceedings of the NationalAcademy of Sciences USA 87, 4645—4645 [sic] (1990)) and transformantswere selected on LB agar containing kanamycin (50 μg/ml) to give the 7.7kb shuttle vector pZ1accDA (FIG. 1). This was incorporated into thestrain DSM5715 by means of electroporation, as described by Haynes (FEMSMicrobiol. Letters 61, 329-334 (1989)), and the transformants wereselected on LBHIS agar (Liebl et al., FEMS Microbiology Letters 65,299-304 (1989)) to give the C. glutamicum strain DSM5715/pZ1accDA.

Example 3

Preparation of L-lysine with the Strain DSM5715/pZ1accDA

After precultivation in medium CgIII (Keilhauer et al., Journal ofBacteriology 175, 5595-5603 (1993)), the strain DSM5715/pZ1accDA wascultivated in production medium CgXII (Keilhauer et al., Journal ofBacteriology 175, 5595-5603 (1993)). 4% of glucose and 50 mg/l ofkanamycin sulfate were added.

After incubation for 48 hours, the optical density at 660 nm and theconcentration of L-lysine formed were determined. The experimentalresults are shown in Table 1.

TABLE 1 L-Lysine Strain OD g/l DSM5175 [sic] 31.4 7.2 DSM5715/pZ1accDA43.1 8.0

Example 4

Joint Expression of accBC and accDA

(i) Construction of expression vector pEK0accBCaccDA Plasmid pWJ71containing accBC (J{umlaut over (ager)} et al., Archives of Microbiology166, 76-82 (1996)) was digested with the restriction enzymes AgeI andSmaI and then treated with Klenow polymerase and alkaline phosphatase.In a parallel operation, plasmid pUCaccDA was digested [sic] EcoRI/XhoIand then treated with Klenow polymerase and alkaline phosphatase. The2.1 kb fragment carrying accDA was isolated by preparative isolationfrom an agarose gel, which was carried out as described by Sambrook etal. (Molecular Cloning, a Laboratory Manual (1989), Cold Spring HarborLaboratories). Said fragment was ligated to vector pWJ71, which had beenprepared as described above. The 4.6 kb fragment carrying accBCaccDA wascleaved from the resulting plasmid by KpnI/SalI digestion and againisolated by preparative agarose gel electrophoresis. To ligate thisfragment to C. glutamicum/E. coli shuttle vector PEK0 (Eikmanns et al.,Gene 102, 93-98 (1991)), pEK0 was digested with the restriction enzymesKpnI and SalI and then treated with Klenow polymerase and alkalinephosphatase. The vector prepared in this way was ligated to the 4.6 kbfragment carrying accBCaccDA. The resulting vector pEK0accBCaccDA isshown in FIG. 2. This vector was incorporated into the strain ATCC13032by means of electroporation (Haynes, FEMS Microbiol. Letters 61, 329-334(1989)), as described in Example 2, to give the C. glutamicum strainATCC13032/pEK0accBCaccDA.

(ii) Determination of the acyl-CoA carboxylase activity After preculturein medium CGIII (Keilhauer et al., Journal of Bacteriology 175,5595-5603 (1993)), the strain C. glutamicum ATCC13032/pEK0accBCaccDA wasgrown in medium CGXII, which is described by Keilhauer et al. (Journalof Bacteriology 175, 5595-5603 (1993)). The cells were harvested bycentrifugation and the cell pellet was washed once with 60 mM Tris-HCl(pH 7.2) and resuspended in the same buffer. The cells were digested bymeans of a 10-minute ultrasound treatment (Branson sonifier W-250,Branson Sonic Power Co., Danbury, USA). The cell debris was thenseparated off by centrifugation for 30 minutes at 4° C. and thesupernatant was used as crude extract in the enzyme test. The reactionmixture for the enzyme test contained 60 mM Tris-HCl (pH 7.2), 65 mMKHCO₃, 1 mM ATP, 1.5 mM MgCl₂, 4 mM acyl-CoA (choice of acetyl-CoA orpropionyl-CoA) and 4 mg of crude extract in a reaction volume of 1 ml.The test mixtures were incubated at 30° C., 100 μl samples were takenafter 15, 30, 45 and 60 seconds and their concentration of malonyl-CoAor methylmalonyl-CoA was determined by means of HPLC analysis (Kimura etal., Journal of Bacteriology 179, 7098-7102 (1997)). As shown in Table2, the strain C. glutamicum ATCC13032/pEK0accBCaccDA exhibits a highacyl-CoA carboxylase activity with both acetyl-CoA and propionyl-CoA,whereas the control strain has only a low acyl-CoA carboxylase activitywith both acetyl-CoA and propionyl-CoA.

Table 2: Specific acyl-CoA carboxylase activity (μmol/min and mgprotein) in C. glutamicum

Acyl-CoA carboxylase activity with the substrate Strain acetyl-CoApropionyl-CoA ATCC13032/pEK0accBCaccDA 0.048 0.124 ATCC13032/pEKO 0.0110.018

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Map of plasmid pZ1accDA

FIG. 2: Map of plasmid pEK0accBCaccDA

3 1 2123 DNA Corynebacterium glutamicum gene (508)..(1980) accDA 1ctcgagcggg agtcggtgat cggccactct ctaagcaatg ccggctttaa aataaagcaa 60cttatatgtt tctcaccaca tctggccgac gaccacgaag tatgttgtcg atcacagcta 120aacgtgtgaa tgtgaagtta cctaactcac attgcaatgc gatagcgatt tggaaaactc 180actcccccca atatcttaac ttaaacttaa aagtagtgtt ttacctgcat ttataaaagt 240tcccgatcta ccccctcttt accccgaaat accccttttg caaagattgc aaacacaaca 300gtgcaatagt taacgggctt cacacgtcac cattctgtcc ggttttaggc tatgttcggg 360acgtctaggc aaaaagtagt tttgtgagat gaaacgcata atccgtcatt ttttacgcaa 420tcgatagcct aaattgggct tagatcttcc gcctctaaat aggtatgcag agacattcga 480attaattaac aaagccattt ttcggccgtg gagaagcgtt ttccgactat ggtgtggggc 540atggaacaca cttcagcatt gacgctcata gactcggttt tggaccctga cagcttcatt 600tcttggaatg aaactcccca atatgacaac ctcaatcaag gctatgcaga gaccttggag 660cgggctcgaa gcaaggccaa atgcgatgaa tcggtaatta ctggagaagg caccgtggag 720ggcattccgg tagccgttat tttgtccgat ttttccttcc tcggcggttc tttgggcacg 780gtcgcgtcgg tgcgcatcat gaaggcgatt caccgcgcca cagagctgaa actcccactg 840ctggtctccc ctgcttccgg tggtgcgcgc atgcaggaag acaatcgagc ttttgtcatg 900atggtgtcca taaccgcggc tgtgcagcgt caccgcgagg cgcatttgcc gttcctggtg 960tatttgcgca atcccacgat gggtggcgcc atggcctcgt ggggttcatc tgggcatctc 1020acttttgcgg aacccggcgc gcagataggt ttcctgggtc ctcgcgtggt ggagttaacc 1080actgggcatg cgcttccaga cggtgtgcag caggcggaga atttggtgaa aactggtgtg 1140attgatggaa ttgtgtcgcc actccaattg cgtgcagcgg tggcaaaaac cctcaaggtt 1200attcagccgg tagaggcaac ggatcgtttt tctccaacaa ctcctggcgt ggcacttccg 1260gtgatggagg cgattgcgcg ttctcgtgac ccgcagaggc ctggaatcgg ggagattatg 1320gaaacgttgg gggcagacgt cgtcaagctt tctggtgcgc gtgctggcgc attgagcccg 1380gctgtgcgcg ttgccctggc gcgcatcggg ggccggcccg tggtgctgat tgggcaggat 1440cgccgcttca cgcttgggcc gcaggagctg cgttttgcgc gtcgtggcat ttcgctggcg 1500cgcgagctaa acctgccgat cgtgtccatc atcgacacct ccggcgccga attgtcgcag 1560gcggctgagg agctcggcat cgcaagctcg attgcgcgca ccttgtccaa gcttatcgac 1620gctcccctcc ccaccgtttc ggtcattatt ggtcagggcg ttggcggtgg cgcgctggcc 1680atgctgcccg ccgatctggt ctacgcggcc gaaaacgcgt ggctgtccgc attgccacca 1740gagggcgcct cggccatcct cttccgcgac accaaccacg ccgcggaaat catagagcga 1800caaggcgtgc aggcgcacgc acttttaagc caagggctta tcgacgggat cgtcgccgaa 1860accgagcact ttgttgaaga aattctcggc acaatcagca acgccctctc cgaattggat 1920aacaatccgg agagggcggg acgcgacagt cgcttcacac gatttgagcg tttagcgcag 1980taaagaaaat tatgcgctga tcaaatcgat gatgaacacc agggtacggc cagacagtgg 2040gtggccggaa ccctcagggc cgtaagcagc ctctggcgga atggtcagct gacgacgtcc 2100gccgaccttc atgcctggaa ttc 2123 2 1473 DNA Corynebacterium glutamicum CDS(1)..(1473) accDA 2 gtg gag aag cgt ttt ccg act atg gtg tgg ggc atg gaacac act tca 48 Val Glu Lys Arg Phe Pro Thr Met Val Trp Gly Met Glu HisThr Ser 1 5 10 15 gca ttg acg ctc ata gac tcg gtt ttg gac cct gac agcttc att tct 96 Ala Leu Thr Leu Ile Asp Ser Val Leu Asp Pro Asp Ser PheIle Ser 20 25 30 tgg aat gaa act ccc caa tat gac aac ctc aat caa ggc tatgca gag 144 Trp Asn Glu Thr Pro Gln Tyr Asp Asn Leu Asn Gln Gly Tyr AlaGlu 35 40 45 acc ttg gag cgg gct cga agc aag gcc aaa tgc gat gaa tcg gtaatt 192 Thr Leu Glu Arg Ala Arg Ser Lys Ala Lys Cys Asp Glu Ser Val Ile50 55 60 act gga gaa ggc acc gtg gag ggc att ccg gta gcc gtt att ttg tcc240 Thr Gly Glu Gly Thr Val Glu Gly Ile Pro Val Ala Val Ile Leu Ser 6570 75 80 gat ttt tcc ttc ctc ggc ggt tct ttg ggc acg gtc gcg tcg gtg cgc288 Asp Phe Ser Phe Leu Gly Gly Ser Leu Gly Thr Val Ala Ser Val Arg 8590 95 atc atg aag gcg att cac cgc gcc aca gag ctg aaa ctc cca ctg ctg336 Ile Met Lys Ala Ile His Arg Ala Thr Glu Leu Lys Leu Pro Leu Leu 100105 110 gtc tcc cct gct tcc ggt ggt gcg cgc atg cag gaa gac aat cga gct384 Val Ser Pro Ala Ser Gly Gly Ala Arg Met Gln Glu Asp Asn Arg Ala 115120 125 ttt gtc atg atg gtg tcc ata acc gcg gct gtg cag cgt cac cgc gag432 Phe Val Met Met Val Ser Ile Thr Ala Ala Val Gln Arg His Arg Glu 130135 140 gcg cat ttg ccg ttc ctg gtg tat ttg cgc aat ccc acg atg ggt ggc480 Ala His Leu Pro Phe Leu Val Tyr Leu Arg Asn Pro Thr Met Gly Gly 145150 155 160 gcc atg gcc tcg tgg ggt tca tct ggg cat ctc act ttt gcg gaaccc 528 Ala Met Ala Ser Trp Gly Ser Ser Gly His Leu Thr Phe Ala Glu Pro165 170 175 ggc gcg cag ata ggt ttc ctg ggt cct cgc gtg gtg gag tta accact 576 Gly Ala Gln Ile Gly Phe Leu Gly Pro Arg Val Val Glu Leu Thr Thr180 185 190 ggg cat gcg ctt cca gac ggt gtg cag cag gcg gag aat ttg gtgaaa 624 Gly His Ala Leu Pro Asp Gly Val Gln Gln Ala Glu Asn Leu Val Lys195 200 205 act ggt gtg att gat gga att gtg tcg cca ctc caa ttg cgt gcagcg 672 Thr Gly Val Ile Asp Gly Ile Val Ser Pro Leu Gln Leu Arg Ala Ala210 215 220 gtg gca aaa acc ctc aag gtt att cag ccg gta gag gca acg gatcgt 720 Val Ala Lys Thr Leu Lys Val Ile Gln Pro Val Glu Ala Thr Asp Arg225 230 235 240 ttt tct cca aca act cct ggc gtg gca ctt ccg gtg atg gaggcg att 768 Phe Ser Pro Thr Thr Pro Gly Val Ala Leu Pro Val Met Glu AlaIle 245 250 255 gcg cgt tct cgt gac ccg cag agg cct gga atc ggg gag attatg gaa 816 Ala Arg Ser Arg Asp Pro Gln Arg Pro Gly Ile Gly Glu Ile MetGlu 260 265 270 acg ttg ggg gca gac gtc gtc aag ctt tct ggt gcg cgt gctggc gca 864 Thr Leu Gly Ala Asp Val Val Lys Leu Ser Gly Ala Arg Ala GlyAla 275 280 285 ttg agc ccg gct gtg cgc gtt gcc ctg gcg cgc atc ggg ggccgg ccc 912 Leu Ser Pro Ala Val Arg Val Ala Leu Ala Arg Ile Gly Gly ArgPro 290 295 300 gtg gtg ctg att ggg cag gat cgc cgc ttc acg ctt ggg ccgcag gag 960 Val Val Leu Ile Gly Gln Asp Arg Arg Phe Thr Leu Gly Pro GlnGlu 305 310 315 320 ctg cgt ttt gcg cgt cgt ggc att tcg ctg gcg cgc gagcta aac ctg 1008 Leu Arg Phe Ala Arg Arg Gly Ile Ser Leu Ala Arg Glu LeuAsn Leu 325 330 335 ccg atc gtg tcc atc atc gac acc tcc ggc gcc gaa ttgtcg cag gcg 1056 Pro Ile Val Ser Ile Ile Asp Thr Ser Gly Ala Glu Leu SerGln Ala 340 345 350 gct gag gag ctc ggc atc gca agc tcg att gcg cgc accttg tcc aag 1104 Ala Glu Glu Leu Gly Ile Ala Ser Ser Ile Ala Arg Thr LeuSer Lys 355 360 365 ctt atc gac gct ccc ctc ccc acc gtt tcg gtc att attggt cag ggc 1152 Leu Ile Asp Ala Pro Leu Pro Thr Val Ser Val Ile Ile GlyGln Gly 370 375 380 gtt ggc ggt ggc gcg ctg gcc atg ctg ccc gcc gat ctggtc tac gcg 1200 Val Gly Gly Gly Ala Leu Ala Met Leu Pro Ala Asp Leu ValTyr Ala 385 390 395 400 gcc gaa aac gcg tgg ctg tcc gca ttg cca cca gagggc gcc tcg gcc 1248 Ala Glu Asn Ala Trp Leu Ser Ala Leu Pro Pro Glu GlyAla Ser Ala 405 410 415 atc ctc ttc cgc gac acc aac cac gcc gcg gaa atcata gag cga caa 1296 Ile Leu Phe Arg Asp Thr Asn His Ala Ala Glu Ile IleGlu Arg Gln 420 425 430 ggc gtg cag gcg cac gca ctt tta agc caa ggg cttatc gac ggg atc 1344 Gly Val Gln Ala His Ala Leu Leu Ser Gln Gly Leu IleAsp Gly Ile 435 440 445 gtc gcc gaa acc gag cac ttt gtt gaa gaa att ctcggc aca atc agc 1392 Val Ala Glu Thr Glu His Phe Val Glu Glu Ile Leu GlyThr Ile Ser 450 455 460 aac gcc ctc tcc gaa ttg gat aac aat ccg gag agggcg gga cgc gac 1440 Asn Ala Leu Ser Glu Leu Asp Asn Asn Pro Glu Arg AlaGly Arg Asp 465 470 475 480 agt cgc ttc aca cga ttt gag cgt tta gcg cag1473 Ser Arg Phe Thr Arg Phe Glu Arg Leu Ala Gln 485 490 3 491 PRTCorynebacterium glutamicum 3 Val Glu Lys Arg Phe Pro Thr Met Val Trp GlyMet Glu His Thr Ser 1 5 10 15 Ala Leu Thr Leu Ile Asp Ser Val Leu AspPro Asp Ser Phe Ile Ser 20 25 30 Trp Asn Glu Thr Pro Gln Tyr Asp Asn LeuAsn Gln Gly Tyr Ala Glu 35 40 45 Thr Leu Glu Arg Ala Arg Ser Lys Ala LysCys Asp Glu Ser Val Ile 50 55 60 Thr Gly Glu Gly Thr Val Glu Gly Ile ProVal Ala Val Ile Leu Ser 65 70 75 80 Asp Phe Ser Phe Leu Gly Gly Ser LeuGly Thr Val Ala Ser Val Arg 85 90 95 Ile Met Lys Ala Ile His Arg Ala ThrGlu Leu Lys Leu Pro Leu Leu 100 105 110 Val Ser Pro Ala Ser Gly Gly AlaArg Met Gln Glu Asp Asn Arg Ala 115 120 125 Phe Val Met Met Val Ser IleThr Ala Ala Val Gln Arg His Arg Glu 130 135 140 Ala His Leu Pro Phe LeuVal Tyr Leu Arg Asn Pro Thr Met Gly Gly 145 150 155 160 Ala Met Ala SerTrp Gly Ser Ser Gly His Leu Thr Phe Ala Glu Pro 165 170 175 Gly Ala GlnIle Gly Phe Leu Gly Pro Arg Val Val Glu Leu Thr Thr 180 185 190 Gly HisAla Leu Pro Asp Gly Val Gln Gln Ala Glu Asn Leu Val Lys 195 200 205 ThrGly Val Ile Asp Gly Ile Val Ser Pro Leu Gln Leu Arg Ala Ala 210 215 220Val Ala Lys Thr Leu Lys Val Ile Gln Pro Val Glu Ala Thr Asp Arg 225 230235 240 Phe Ser Pro Thr Thr Pro Gly Val Ala Leu Pro Val Met Glu Ala Ile245 250 255 Ala Arg Ser Arg Asp Pro Gln Arg Pro Gly Ile Gly Glu Ile MetGlu 260 265 270 Thr Leu Gly Ala Asp Val Val Lys Leu Ser Gly Ala Arg AlaGly Ala 275 280 285 Leu Ser Pro Ala Val Arg Val Ala Leu Ala Arg Ile GlyGly Arg Pro 290 295 300 Val Val Leu Ile Gly Gln Asp Arg Arg Phe Thr LeuGly Pro Gln Glu 305 310 315 320 Leu Arg Phe Ala Arg Arg Gly Ile Ser LeuAla Arg Glu Leu Asn Leu 325 330 335 Pro Ile Val Ser Ile Ile Asp Thr SerGly Ala Glu Leu Ser Gln Ala 340 345 350 Ala Glu Glu Leu Gly Ile Ala SerSer Ile Ala Arg Thr Leu Ser Lys 355 360 365 Leu Ile Asp Ala Pro Leu ProThr Val Ser Val Ile Ile Gly Gln Gly 370 375 380 Val Gly Gly Gly Ala LeuAla Met Leu Pro Ala Asp Leu Val Tyr Ala 385 390 395 400 Ala Glu Asn AlaTrp Leu Ser Ala Leu Pro Pro Glu Gly Ala Ser Ala 405 410 415 Ile Leu PheArg Asp Thr Asn His Ala Ala Glu Ile Ile Glu Arg Gln 420 425 430 Gly ValGln Ala His Ala Leu Leu Ser Gln Gly Leu Ile Asp Gly Ile 435 440 445 ValAla Glu Thr Glu His Phe Val Glu Glu Ile Leu Gly Thr Ile Ser 450 455 460Asn Ala Leu Ser Glu Leu Asp Asn Asn Pro Glu Arg Ala Gly Arg Asp 465 470475 480 Ser Arg Phe Thr Arg Phe Glu Arg Leu Ala Gln 485 490

What is claimed is:
 1. An isolated DNA consisting essentially ofnucleotides encoding a protein having the amino acid sequence of SEQ IDNO:3.
 2. The isolated DNA of claim 1, wherein said DNA has thenucleotide sequence of SEQ ID NO:1.
 3. The isolated DNA of claim 1,wherein said DNA has the nucleotide sequence of SEQ ID NO:2.
 4. Theplasmid pZ1accDA deposited in C. glutamicum under deposit number DSM12785.
 5. A vector for expressing a protein with accDA activity, saidvector comprising a coding region consisting of the DNA of any one ofclaims 1-3.
 6. A C. glutamicum bacterium transformed with the vector ofclaim 5.